Improving preclinical to clinical translation in Alzheimer\u27s disease research.

Introduction: Preclinical testing in animal models is a critical component of the drug discovery and development process. While hundreds of interventions have demonstrated preclinical efficacy for ameliorating cognitive impairments in animal models, none have confirmed efficacy in Alzheimer\u27s disease (AD) clinical trials. Critically this lack of translation to the clinic points in part to issues with the animal models, the preclinical assays used, and lack of scientific rigor and reproducibility during execution. In an effort to improve this translation, the Preclinical Testing Core (PTC) of the Model Organism Development and Evaluation for Late-onset AD (MODEL-AD) consortium has established a rigorous screening strategy with go/no-go decision points that permits unbiased assessments of therapeutic agents. Methods: An initial screen evaluates drug stability, formulation, and pharmacokinetics (PK) to confirm appreciable brain exposure in the disease model at the pathologically relevant ages, followed by pharmacodynamics (PD) and predictive PK/PD modeling to inform the dose regimen for long-term studies. The secondary screen evaluates target engagement and disease modifying activity using non-invasive positron emission tomography/magnetic resonance imaging (PET/MRI). Provided the compound meets its go criteria for these endpoints, evaluation for efficacy on behavioral endpoints are conducted. Results: Validation of this pipeline using tool compounds revealed the importance of critical quality control (QC) steps that researchers need to be aware of when executing preclinical studies. These include confirmation of the active pharmaceutical ingredient and at the precise concentration expected; and an experimental design that is well powered and in line with the Animal Research Reporting of In vivo Experiments (ARRIVE) guidelines. Discussion: Taken together our experience executing a rigorous screening strategy with QC checkpoints provides insight to the challenges of conducting translational studies in animal models. The PTC pipeline is a National Institute on Aging (NIA)-supported resource accessible to the research community for investigators to nominate compounds for testing (https://stopadportal.synapse.org/), and these resources will ultimately enable better translational studies to be conducted

In Vivo Targeting Replication Protein A for Cancer Therapy

Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination, and the DNA damage response (DDR). Retrospective analysis of lung cancer patient data demonstrates high RPA expression as a negative prognostic biomarker for overall survival in smoking-related lung cancers. Similarly, relative expression of RPA is a predictive marker for response to chemotherapy. These observations are consistent with the increase in RPA expression serving as an adaptive mechanism that allows tolerance of the genotoxic stress resulting from carcinogen exposure. We have developed second-generation RPA inhibitors (RPAis) that block the RPA-DNA interaction and optimized formulation for in vivo analyses. Data demonstrate that unlike first-generation RPAis, second-generation molecules show increased cellular permeability and induce cell death via apoptosis. Second-generation RPAis elicit single-agent in vitro anticancer activity across a broad spectrum of cancers, and the cellular response suggests existence of a threshold before chemical RPA exhaustion induces cell death. Chemical RPA inhibition potentiates the anticancer activity of a series of DDR inhibitors and traditional DNA-damaging cancer therapeutics. Consistent with chemical RPA exhaustion, we demonstrate that the effects of RPAi on replication fork dynamics are similar to other known DDR inhibitors. An optimized formulation of RPAi NERx 329 was developed that resulted in single-agent anticancer activity in two non-small cell lung cancer models. These data demonstrate a unique mechanism of action of RPAis eliciting a state of chemical RPA exhaustion and suggest they will provide an effective therapeutic option for difficult-to-treat lung cancers

Prophylactic evaluation of verubecestat on disease- and symptom-modifying effects in 5XFAD mice.

Introduction: Alzheimer\u27s disease (AD) is the most common form of dementia. Beta-secretase (BACE) inhibitors have been proposed as potential therapeutic interventions; however, initiating treatment once disease has significantly progressed has failed to effectively stop or treat disease. Whether BACE inhibition may have efficacy when administered prophylactically in the early stages of AD has been under-investigated. The present studies aimed to evaluate prophylactic treatment of the BACE inhibitor verubecestat in an AD mouse model using the National Institute on Aging (NIA) resources of the Model Organism Development for Late-Onset Alzheimer\u27s Disease (MODEL-AD) Preclinical Testing Core (PTC) Drug Screening Pipeline. Methods: 5XFAD mice were administered verubecestat ad libitum in chow from 3 to 6 months of age, prior to the onset of significant disease pathology. Following treatment (6 months of age), in vivo imaging was conducted with 18F-florbetapir (AV-45/Amyvid) (18F-AV45) and 18-FDG (fluorodeoxyglucose)-PET (positron emission tomography)/MRI (magnetic resonance imaging), brain and plasma amyloid beta (Aβ) were measured, and the clinical and behavioral characteristics of the mice were assessed and correlated with the pharmacokinetic data. Results: Prophylactic verubecestat treatment resulted in dose- and region-dependent attenuations of 18F-AV45 uptake in male and female 5XFAD mice. Plasma Aβ40 and Aβ42 were also dose-dependently attenuated with treatment. Across the dose range evaluated, side effects including coat color changes and motor alterations were reported, in the absence of cognitive improvement or changes in 18F-FDG uptake. Discussion: Prophylactic treatment with verubecestat resulted in attenuated amyloid plaque deposition when treatment was initiated prior to significant pathology in 5XFAD mice. At the same dose range effective at attenuating Aβ levels, verubecestat produced side effects in the absence of improvements in cognitive function. Taken together these data demonstrate the rigorous translational approaches of the MODEL-AD PTC for interrogating potential therapeutics and provide insight into the limitations of verubecestat as a prophylactic intervention for early-stage AD

New Ref-1/APE1 targeted inhibitors demonstrating improved potency for clinical applications in multiple cancer types

AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein that is overexpressed in most aggressive cancers and impacts various cancer cell signaling pathways. Ref-1′s redox activity plays a significant role in activating transcription factors (TFs) such as NFκB, HIF1α, STAT3 and AP-1, which are crucial contributors to the development of tumors and metastatic growth. Therefore, development of potent, selective inhibitors to target Ref-1 redox function is an appealing approach for therapeutic intervention. A first-generation compound, APX3330 successfully completed phase I clinical trial in adults with progressing solid tumors with favorable response rate, pharmacokinetics (PK), and minimal toxicity. These positive results prompted us to develop more potent analogs of APX3330 to effectively target Ref-1 in solid tumors. In this study, we present structure-activity relationship (SAR) identification and validation of lead compounds that exhibit a greater potency and a similar or better safety profile to APX3330. In order to triage and characterize the most potent and on-target second-generation Ref-1 redox inhibitors, we assayed for PK, mouse and human S9 fraction metabolic stability, in silico ADMET properties, ligand-based WaterLOGSY NMR measurements, pharmacodynamic markers, cell viability in multiple cancer cell types, and two distinct 3-dimensional (3D) cell killing assays (Tumor-Microenvironment on a Chip and 3D spheroid). To characterize the effects of Ref-1 inhibition in vivo, global proteomics was used following treatment with the top four analogs. This study identified and characterized more potent inhibitors of Ref-1 redox function (that outperformed APX3330 by 5–10-fold) with PK studies demonstrating efficacious doses for translation to clinic

Prophylactic evaluation of verubecestat on disease‐ and symptom‐modifying effects in 5XFAD mice

Abstract Introduction Alzheimer's disease (AD) is the most common form of dementia. Beta‐secretase (BACE) inhibitors have been proposed as potential therapeutic interventions; however, initiating treatment once disease has significantly progressed has failed to effectively stop or treat disease. Whether BACE inhibition may have efficacy when administered prophylactically in the early stages of AD has been under‐investigated. The present studies aimed to evaluate prophylactic treatment of the BACE inhibitor verubecestat in an AD mouse model using the National Institute on Aging (NIA) resources of the Model Organism Development for Late‐Onset Alzheimer's Disease (MODEL‐AD) Preclinical Testing Core (PTC) Drug Screening Pipeline. Methods 5XFAD mice were administered verubecestat ad libitum in chow from 3 to 6 months of age, prior to the onset of significant disease pathology. Following treatment (6 months of age), in vivo imaging was conducted with 18F‐florbetapir (AV‐45/Amyvid) (18F‐AV45) and 18‐FDG (fluorodeoxyglucose)–PET (positron emission tomography)/MRI (magnetic resonance imaging), brain and plasma amyloid beta (Aβ) were measured, and the clinical and behavioral characteristics of the mice were assessed and correlated with the pharmacokinetic data. Results Prophylactic verubecestat treatment resulted in dose‐ and region‐dependent attenuations of 18F‐AV45 uptake in male and female 5XFAD mice. Plasma Aβ40 and Aβ42 were also dose‐dependently attenuated with treatment. Across the dose range evaluated, side effects including coat color changes and motor alterations were reported, in the absence of cognitive improvement or changes in 18F‐FDG uptake. Discussion Prophylactic treatment with verubecestat resulted in attenuated amyloid plaque deposition when treatment was initiated prior to significant pathology in 5XFAD mice. At the same dose range effective at attenuating Aβ levels, verubecestat produced side effects in the absence of improvements in cognitive function. Taken together these data demonstrate the rigorous translational approaches of the MODEL‐AD PTC for interrogating potential therapeutics and provide insight into the limitations of verubecestat as a prophylactic intervention for early‐stage AD